molecules
Article
Towards Water Soluble Mitochondria-Targeting
Theranostic Osmium(II) Triazole-Based Complexes
Salem A. E. Omar 1 , Paul A. Scattergood 1 , Luke K. McKenzie 2,3 , Helen E. Bryant 3 ,
Julia A. Weinstein 2 and Paul I. P. Elliott 1, *
1
2
3
*
Department of Chemistry, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK;
[email protected] (S.A.E.O.); [email protected] (P.A.S.)
Department of Chemistry, Dainton Building, University of Sheffield, Sheffield S3 7HF, UK;
[email protected] (L.K.M.); [email protected] (J.A.W.)
Academic Unit of Molecular Oncology, Sheffield Institute for Nucleic Acids (SInFoNiA),
Department of Oncology and Metabolism, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK;
[email protected]
Correspondence: [email protected]; Tel.: +44-1484-472320
Academic Editor: James Crowley
Received: 29 September 2016; Accepted: 12 October 2016; Published: 18 October 2016
Abstract: The complex [Os(btzpy)2 ][PF6 ]2 (1, btzpy = 2,6-bis(1-phenyl-1,2,3-triazol-4-yl)pyridine) has
been prepared and characterised. Complex 1 exhibits phosphorescence (λem = 595 nm, τ = 937 ns,
φem = 9.3% in degassed acetonitrile) in contrast to its known ruthenium(II) analogue, which is
non-emissive at room temperature. The complex undergoes significant oxygen-dependent quenching
of emission with a 43-fold reduction in luminescence intensity between degassed and aerated
acetonitrile solutions, indicating its potential to act as a singlet oxygen sensitiser. Complex 1
underwent counterion metathesis to yield [Os(btzpy)2 ]Cl2 (1Cl ), which shows near identical optical
absorption and emission spectra to those of 1. Direct measurement of the yield of singlet oxygen
sensitised by 1Cl was carried out (φ (1 O2 ) = 57%) for air equilibrated acetonitrile solutions. On the
basis of these photophysical properties, preliminary cellular uptake and luminescence microscopy
imaging studies were conducted. Complex 1Cl readily entered the cancer cell lines HeLa and U2OS
with mitochondrial staining seen and intense emission allowing for imaging at concentrations as
low as 1 µM. Long-term toxicity results indicate low toxicity in HeLa cells with LD50 >100 µM.
Osmium(II) complexes based on 1 therefore present an excellent platform for the development of
novel theranostic agents for anticancer activity.
Keywords: triazole; osmium; photophysics; complexes; ligands; anticancer; oxygen sensitizer
1. Introduction
Oligopyridyl complexes of kinetically inert d6 metals, e.g., Ru(II), Os(II), have attracted enormous
interest in recent decades due to their attractive photophysical properties [1–4]. These complexes
typically exhibit relatively long-lived triplet metal-to-ligand charge transfer (3 MLCT) states.
These states may undergo deactivation through a number of routes including phosphorescence or
energy/electron transfer which enables the potential application of these complexes in light-emitting [5]
and photovoltaic technologies [6]. Key to the development of complexes for these applications is the
design of the ligands supporting these metals. We, and others, have paid particular attention to the
use of copper-catalysed coupling of alkynes and azides to form 1,2,3-triazole-based ligands [7–10] and
have investigated the photophysical properties of their resultant complexes. A significant number of
reports have appeared detailing the photophysical and photochemical properties of triazole-based
complexes of Re(I), Ru(II) and Ir(III) [11–25]. Examples of triazole-containing complexes of osmium(II)
Molecules 2016, 21, 1382; doi:10.3390/molecules21101382
www.mdpi.com/journal/molecules
Molecules 2016, 21, 1382
2 of 12
are, however, comparatively rare. We have recently reported the synthesis and characterization of
deep-red/near-IR emissive osmium(II) bitriazolyl (btz) complexes and demonstrated their use in
light-emitting electrochemical cells [19].
Molecules 2016, 21, 1382
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Of recent and growing interest has been the use of luminescent complexes in biological
applications
[26–28].
phosphorescence
is longer-lived
thanosmium(II)
the autofluorescence
reported the
synthesisSince
and characterization
of deep-red/near-IR
emissive
bitriazolyl (btz)from
biological
organic
compounds, their
emissive
complexes are
amenable to
use
in time-gated imaging
complexes
and demonstrated
use in light-emitting
electrochemical
cells
[19].
Of [29–31].
recent and
growing complexes,
interest has however,
been the often
use ofexhibit
luminescent
complexes
in biologicaldirect
microscopy
Osmium(II)
lower energy
spin-forbidden
3 MLCT
applications
[26–28].
Since
phosphorescence
is
longer-lived
than
the
autofluorescence
from
biological
optical absorption bands of moderate extinction coefficient due to the high spin-orbit
coupling
organic
compounds,
emissive
complexes
are
amenable
to
use
in
time-gated
imaging
microscopy
[29–
constant for the osmium centre [32,33]. This offers the advantage of enabling efficient excitation
31]. Osmium(II) complexes, however, often exhibit lower energy spin-forbidden direct 3MLCT optical
at lower energies that therefore avoids potential cellular damage and negates autofluorescence
absorption bands of moderate extinction coefficient due to the high spin-orbit coupling constant for
and the necessity of the added expense of time-gated apparatus. These absorption and emission
the osmium centre [32,33]. This offers the advantage of enabling efficient excitation at lower energies
bandsthat
at therefore
wavelengths
closer to the red in comparison to those of common iridium(III) complexes,
avoids potential cellular damage and negates autofluorescence and the necessity of the
and therefore
in
a
more
biologically
transparent
region and
of the
spectrum,
will
also enable
greater
added expense of time-gated
apparatus.
These absorption
emission
bands at
wavelengths
closer
depthtoofthe
penetration
for excitation
andofimaging.
of osmium(II)
also typically
highly
red in comparison
to those
common Complexes
iridium(III) complexes,
and are
therefore
in a more
inert biologically
to ligand photosubstitution
making
them highly
robust
[34]
(although
thepenetration
unprecedentedly
transparent region of
the spectrum,
will also
enable
greater
depth of
for
2+ wasalso
and imaging.inComplexes
of [Os(btz)
osmium(II)
typically
highly
inertThe
to intensity
ligand of
facileexcitation
ligand photoejection
the complex
recently
reported
[35]).
3 ] are
photosubstitution
making
them
highly
robust
[34]
(although
the
unprecedentedly
facile
ligand
phosphorescence is, however, sensitive to the presence of oxygen resulting in quenching
through
2+
3
1
photoejection
in state
the complex
[Os(btz)O
3]
was enabling
recently exploitation
reported [35]).
The intensity therapy
of
conversion
of ground
O2 to reactive
,
thus
in
photodynamic
2
phosphorescence is, however, sensitive to the presence of oxygen resulting in quenching through
(PDT) [36]. These combined properties thus present significant opportunities for the development of
conversion of ground state 3O2 to reactive 1O2, thus enabling exploitation in photodynamic therapy
unique dual-mode theranostic agents.
(PDT) [36]. These combined properties thus present significant opportunities for the development of
We
report here the synthesis and characterization of the orange-emissive bis(terdentate)
unique dual-mode theranostic agents.
2+ (btzpy = 2,6-bis(1-phenyl-1,2,3-triazol-4-yl)pyridine) as its
osmium(II)
[Os(btzpy)
2]
We complex
report here
the synthesis
and characterization of the orange-emissive bis(terdentate)
hexafluorophosphate
(1) [Os(btzpy)
and chloride
(1Cl ) salts.
The complex shows significant dependence
osmium(II) complex
2]2+ (btzpy
= 2,6-bis(1-phenyl-1,2,3-triazol-4-yl)pyridine)
as its of
Cl
Cl
emission
intensity on the presence
of oxygen.
water
salt 1 hasdependence
been subjected
hexafluorophosphate
(1) and chloride
(1 )The
salts.
The soluble
complexchloride
shows significant
of to
Cl has been subject to
emissioncellular
intensity
on theand
presence
of oxygen.imaging
The waterstudies
solubleand
chloride
salt 1results
preliminary
uptake
luminescence
relevant
are reported.
preliminary cellular uptake and luminescence imaging studies and relevant results are reported.
2. Results & Discussion
2. Results & Discussion
Complex 1 was prepared by reaction of two equivalents of the ligand btzpy [37,38] with
Complex 1 was prepared by reaction of two equivalents of the ligand btzpy [37,38] with
[OsCl6 ][NH
4 ]2 in refluxing ethylene glycol (Scheme 1). After being left to cool, the complex
[OsCl6][NH4]2 in refluxing ethylene glycol (Scheme 1). After being left to cool, the complex was
was isolated as an orange powder, its hexafluorophosphate salt, through treatment with NH
PF .
isolated as an orange powder, its hexafluorophosphate salt, through treatment with NH4PF6. The 1H- 4 6
1
The NMR
H-NMR
spectrum of 1 exhibits a characteristic singlet resonance for four equivalent triazole
spectrum of 1 exhibits a characteristic singlet resonance for four equivalent triazole ring protons
ring at
protons
at
δ is9.13,
whichrelative
is deshielded
relative to
the for
corresponding
signal
for The
the free
δ 9.13, which
deshielded
to the corresponding
signal
the free ligand by
0.16 ppm.
ligand
by 0.16
ppm.
The
protons
therise
central
pyridine
ring give
rise toatdoublet
and
triplet
protons
of the
central
pyridine
ringofgive
to doublet
and triplet
resonances
δ 8.36 and
8.01,
resonances
at δ with
8.36 those
and 8.01,
with those
forinthe
phenylbetween
substituents
resulting
respectively,
for therespectively,
phenyl substituents
resulting
multiplets
δ 7.50 and
7.60. in
For spectroscopic
comparison
theFor
complex
[Os(tolterpy)
2][PF6]2 (2,
tolterpy
= 4′-p-tolyl-2,2′:6′,2′′multiplets
between δ 7.50
and 7.60.
spectroscopic
comparison
the
complex
[Os(tolterpy)2 ][PF6 ]2
0
0
0
00
terpyridine)
was
also
prepared.
(2, tolterpy = 4 -p-tolyl-2,2 :6 ,2 -terpyridine) was also prepared.
Scheme
1. 1.Synthesis
Scheme
Synthesisof
of [Os(btzpy)
[Os(btzpy)2][PF
6]26 ]
(1).
2 ][PF
2 (1).
The electrochemical properties of 1 were investigated by cyclic voltammetry (CV) and reveal a
The electrochemical
properties of 1 were investigated by cyclic voltammetry (CV) and reveal
reversible Os(II)/Os(III) oxidation at +0.64 V (vs Fc/Fc+ = 0.0+V). This is close to that exhibited by the
a reversible Os(II)/Os(III) oxidation
at +0.64 V (vs Fc/Fc = 0.0 V). This is close to that exhibited
known model complex 2 (Eox = +0.49
V) and other related osmium(II) complexes [39–41] indicating
ox =
by the
known
model
complex
2
(E
+0.49 V) and other related osmium(II) complexes [39–41]
that the highest occupied molecular orbital (HOMO) has primarily metallic 5d orbital character.
indicating
theCV
highest
occupied
molecular reductions
orbital (HOMO)
has primarily
metallic
5d orbital
Unlikethat
in the
trace for
2, no ligand-based
are observed
for 1 within
the available
Molecules 2016, 21, 1382
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character. Unlike in the CV trace for 2, no ligand-based reductions are observed for 1 within the
Molecules 2016, 21, 1382
3 of 12
available
electrochemical window (−2.0 to +1.2 V), which is indicative of the higher energy
lowest
unoccupied molecular orbital (LUMO) localized on the btzpy ligand compared to that for the tolterpy
electrochemical window (−2.0 to +1.2 V), which is indicative of the higher energy lowest unoccupied
complex.
This LUMO
destabilisation
consistent
previously
on the ruthenium(II)
molecular
orbital (LUMO)
localizedison
the btzpywith
ligand
compared reported
to that fordata
the tolterpy
complex.
2+ (terpy = 2,20 :60 ,200 -terpyridine) [42–44].
analogue
of
1
versus
[Ru(terpy)
]
This LUMO destabilisation is 2consistent with previously reported data on the ruthenium(II) analogue
UV-visible
absorption
spectra
were recorded for[42–44].
1 and 2 in acetonitrile solutions at room
of 1 versus [Ru(terpy)
2]2+ (terpy
= 2,2′:6′,2′′-terpyridine)
UV-visible
spectra
forof1 1and
2 in acetonitrile
solutions at atroom
temperature
(Figureabsorption
1a and Table
1). were
The recorded
spectrum
exhibits
a strong absorption
297 nm
temperature
(Figure
1a
and
Table
1).
The
spectrum
of
1
exhibits
a
strong
absorption
at
297
nmband
assigned to ligand-centred π→π* transitions localized on the btzpy ligand along with a broad
1
assigned
to
ligand-centred
π→π*
transitions
localized
on
the
btzpy
ligand
along
with
a
broad
band
between 350 and 400 nm assigned to MLCT transitions. This is significantly blue-shifted relative
between
350 and
nm assigned
to 1MLCT
transitions.
Thisligand,
is significantly
blue-shifted
relativehaving
to
to that
observed
for 400
2 (491
nm) consistent
with
the btzpy
and hence
its complex,
that observed for 2 (491 nm) consistent with the btzpy ligand, and hence its complex, having a much
a much higher energy LUMO as indicated from the CV data described above. Similarly to the data
higher energy LUMO as indicated from the CV data described above. Similarly to the data for 2,
for 2, complex
complex1 1also
also
exhibits absorptions of lesser intensity at longer wavelengths corresponding to
exhibits absorptions of lesser intensity at longer wavelengths corresponding to spin3 MLCT excitations (λmax = 526 nm) from the singlet ground state enabled by the
spin-forbidden
direct
forbidden direct 3MLCT
excitations (λmax = 526 nm) from the singlet ground state enabled by the large
large spin-orbit
spin-orbitcoupling
coupling
constant
associated
the osmium
constant
associated
withwith
the osmium
centre centre
[32,33].[32,33].
(a)
(b)
Figure 1. (a) UV-visible absorption spectra for 1 and 2 in acetonitrile solutions and 1Cl in aqueous
Figure 1. (a) UV-visible absorption spectra for 1 and 2 in acetonitrile solutions and 1Cl in aqueous
solution at room temperature; (b) normalized emission spectra of complexes 1 and 2 in de-aerated
solution at room temperature; Cl(b) normalized emission spectra of complexes 1 and 2 in de-aerated
acetonitrile solutions and 1 in aerated aqueous solution at room temperature (solid lines) and
Cl in aerated aqueous solution at room temperature (solid lines) and
acetonitrile
solutions
and
complexes
1 and 2 in
4:11EtOH/MeOH
glasses at 77 K (dashed lines).
complexes 1 and 2 in 4:1 EtOH/MeOH glasses at 77 K (dashed lines).
Table 1. Summarised photophysical data for 1, 1Cl and 2 in acetonitrile.
Complex
1
Complex
1 1Cl
Table 1.absSummarised
photophysical
data forem1, 1Cl1 and 2 in2,3acetonitrile.2,4
1
3
−1
−1
λ /nm (ε/dm ·mol ·cm )
λ /nm
τ/ns
526 (3025), 434 (5700), 382 (19,500),
6
2,3 ± 12
1 (ε/dm3 ·mol−1 ·cm−1 )
λem /nm 1 595 τ/ns 937
λabs337
/nm(13,500),
297 (68,500), 288 (49,000)
534 (3315),
438 (5800),
390 (24,750),
526 (3025),
434 (5700),
382 (19,500),
595 6
337 (13,500),
297 (68,500),
288 (49,000)
345 (17,350),
297 (90,800),
287 (62,500)
φem/%
λem/nm 5
φem9.3
/% 2,4
884 ± 6
599 6
937 ± 12
(273 ± 3) 8
(589) 6,8
6
564,
λem606
/nm 5
9.7
9.3
(5.4) 8
564,- 606 6
669(3315),
(5070),438
645(5800),
(4600),390
491(24,750),
(1930), 406 (7520),
534
6 ±4
9.7 3.2
(5.4)
599 6
718, 795
738 7 884 ±339
- 7
8
345 (17,350), 314
297 (54,300),
(90,800), 286
287 (48,000)
(62,500)
(273 ± 3) 8
(589) 6,8
1 RT, acetonitrile solutions; 2 Degassed MeCN at RT; 3 λex = 405 nm; 4 Relative to [Ru(bpy)3][PF6]2 φem =
669 (5070), 645 (4600), 491 (1930), 406
2
±4
3.2
738 7glass; 6 λex =339
718, 795 7
5 77 K, 4:1 EtOH/MeOH
500 nm; 7 λex = 600 nm; 8 Aerated
0.018 in aerated
MeCN
[45]; 286
(7520), 314
(54,300),
(48,000)
solution.
1 RT,aqueous
acetonitrile
solutions; 2 Degassed MeCN at RT; 3 λex = 405 nm; 4 Relative to [Ru(bpy) ][PF ] φ = 0.018 in
1Cl 2
3
6 2
em
aerated MeCN [45]; 5 77 K, 4:1 EtOH/MeOH glass; 6 λex = 500 nm; 7 λex = 600 nm; 8 Aerated aqueous solution.
In stark contrast to its ruthenium(II) analogue, 1 is emissive at room temperature in de-aerated
acetonitrile solutions, with the emission being characterized by a broad featureless band at 595 nm
In
its ruthenium(II)
analogue,
is emissive
at room
temperature
in de-aerated
= 500contrast
nm) and to
a lifetime
of 937 ns (Figure
1b and1Table
1) attributed
to an
emissive 3MLCT
state.
(λexstark
Bis(tridentate)
complexes
of ruthenium(II)
typically
show littleby
or anobroad
emission
at room temperature
acetonitrile
solutions,
with the
emission being
characterized
featureless
band at 595 nm
thenm)
deviation
an ideal
octahedral-like
coordination
resultstoinan
stabilization
tripletstate.
(λex =as500
and afrom
lifetime
of 937
ns (Figure 1b
and Tablegeometry
1) attributed
emissive 3of
MLCT
3MC) states relative to the 3MLCT state [46,47]. As such, 3MC states are efficiently
metal-centred
(
Bis(tridentate) complexes of ruthenium(II) typically show little or no emission at room temperature
populated from photoexcited 3MLCT states thereby quenching emission. The observed emission for
as the deviation from an ideal octahedral-like coordination
geometry results in stabilization of triplet
1 must therefore
arise from the destabilization
of the 3MC states due to the 3typically larger ligand3
3
metal-centred ( MC) states relative to the MLCT state [46,47]. As such, MC states are efficiently
field splitting associated with
the 5d metal centre over its 4d counterpart, such that non-radiative
3
populated
from photoexcited
quenching
emission.
The observed
emission
3MLCTstates
state isthereby
comparatively
disfavoured.
Mirroring
the UV-visible
depopulation
of the emissiveMLCT
3
for 1 absorption
must therefore
arise
from spectra
the destabilization
of theblue-shifted
MC states
due to
the typically
larger
data, the
emission
of 1 are significantly
relative
to those
of 2 by 143
3MLCT
ligand-field
splitting
associated
the 5d metal centre
over its
4d counterpart,
suchalso
thatits
non-radiative
nm (3260
cm−1) indicative
of with
the comparatively
destabilized
LUMO
for 1, and hence
depopulation of the emissive 3 MLCT state is comparatively disfavoured. Mirroring the UV-visible
Molecules 2016, 21, 1382
Molecules 2016, 21, 1382
4 of 12
4 of 12
state. The emission spectrum was also recorded at 77 K in a 4:1 EtOH/MeOH glass matrix and shows
aabsorption
structured
emission
band that
is blue-shifted
relative toblue-shifted
the spectrum
at room
temperature
due
to
Moleculesdata,
2016,
21,
4 of143
12 nm
the1382
emission
spectra
of 1 are significantly
relative
to those
of 2 by
rigidochromic
effects.
−
1
3
(3260 cm ) indicative of the comparatively destabilized LUMO for 1, and hence also its MLCT state.
state. The emission
was
also recordedaffected
at 77 K inby
a 4:1
EtOH/MeOH
glass
matrix
and shows
Emission
intensityspectrum
from
1 is
dramatically
the
presence ofglass
oxygen
(Figure
2)
and isa
The emission
spectrum
was
also
recorded
at 77relative
K in a to
4:1the
EtOH/MeOH
matrix
and
shows
a
structured
emission
band
that
is
blue-shifted
spectrum
at
room
temperature
due
to
quenched
by
approximately
43-fold
when recorded
conditions.
The
structured
emission
band that
is blue-shifted
relativeintoair
thecompared
spectrumtoatdeaerated
room temperature
due
to
rigidochromic
effects.
3MLCTlong
lifetime
of
emission
combined
with
the
oxygen
sensitivity
confirms
the
assignment
of
a
rigidochromic
effects.
Emission intensity from 1 is dramatically affected by the presence of oxygen (Figure 2) and is
basedEmission
emissiveintensity
state. This
significant
quenchingaffected
of emission
bypresence
oxygen thus
presents
the possibility
from
1 is
dramatically
by compared
the
of oxygen
(Figure
2)
and is
quenched by
approximately
43-fold
when recorded in air
to deaerated
conditions.
The
1O2 sensitizers for photodynamic therapeutic
of
utilizing
complexes
based
on
1
as
potential
long lifetime
of emission combined
with recorded
the oxygeninsensitivity
confirms
the assignment
of a 3MLCTquenched
by approximately
43-fold when
air compared
to deaerated
conditions.
The long
applications.
3 MLCT-based
based
emissive state.
This significant
of emission
by oxygen
presents of
thea possibility
lifetime
of emission
combined
with the quenching
oxygen sensitivity
confirms
the thus
assignment
of utilizing
complexes
basedquenching
on 1 as potential
O2 sensitizers
photodynamic
emissive
state. This
significant
of emission
by oxygenforthus
presents thetherapeutic
possibility of
applications.
1
utilizing complexes based on 1 as potential O2 sensitizers for photodynamic therapeutic applications.
1
Figure 2. (a)
(a) Relative
Relative oxygen
oxygen dependent
dependent emission
emission spectra
spectra for
for 11 in
in acetonitrile
acetonitrile (normalized
(normalized for
Figure 2. (a) Relative oxygen dependent emission spectra for 1 in acetonitrile (normalized for
0 and
I
are
the
emission
intensities
at
deoxygenated
deoxygenated (vacuum)
(vacuum)conditions)
conditions)and
and(b)
(b)Stern-Volmer
Stern-Volmerplot
plot(I(I
and
I
are
the
emission
intensities
0
deoxygenated (vacuum) conditions) and (b) Stern-Volmer plot (I0 and I are the emission intensities at
λatmax
for for
thethe
degassed
solution
and
solution
at at
the
λmax
degassed
solution
and
solution
thepartial
partialpressure
pressureofofoxygen
oxygenat
at which
which emission
emission is
λmax for the degassed solution and solution at the partial pressure of oxygen at which emission is
measured
respectively).
measured
respectively).
Complex
was
alsostudied
studied
functional
theory
(DFT)
calculations
to confirm
the
Complex
1 also
was
also
studied
bydensity
density
functional
theory
(DFT)
calculations
to confirm
Complex
11 was
byby
density
functional
theory
(DFT)
calculations
to confirm
thethe
nature,
nature,
localisation
and
relative
energies
of
the
frontier
orbitals
as
well
as
to
simulate
the
optical
nature,
localisation
and
relative
energies
of
the
frontier
orbitals
as
well
as
to
simulate
the
optical
localisation and relative energies of the frontier orbitals as well as to simulate the optical absorption
absorption
spectrum.
data
reveal
thatthe
theHOMO
HOMO is
localized
primarily
onon
thethe
osmium(II)
centre
absorption
spectrum.
TheThe
data
reveal
that
is
localizedon
primarily
osmium(II)
centre
spectrum. The
data reveal
that the
HOMO
is localized
primarily
the osmium(II)
centre as expected
as
expected
(Figure
3a)
but
with
a
small
contribution
from
the
π-systems
of
the
four
triazole
rings.rings.
as
expected
(Figure
3a)
but
with
a
small
contribution
from
the
π-systems
of
the
four
triazole
(Figure 3a) but with a small contribution from the π-systems of the four triazole rings. The LUMO is
The LUMO
is localized
the
btzpyligands,
ligands, predominantly
predominantly onon
the
central
pyridine ring ring
and and
The
LUMO
isone
localized
on on
oneone
of of
the
btzpy
the
central
localized
of the btzpy
ligands,
predominantly
pyridine
ringpyridine
and
with a lesser
with on
a lesser
contribution
from
the triazole
rings (Figureon
3b)the
butcentral
also a metallic
d-orbital
contribution.
with
a lesser from
contribution
from the triazole
rings
(Figure
3b) but also
a metallic
d-orbitalThe
contribution.
contribution
(Figure
3b)
but
a metallic
contribution.
HOMO
The HOMO ofthe
1 istriazole
slightlyrings
stabilized
(−10.63
eV) also
relative
to that d-orbital
of 2 (−10.35
eV) in agreement
with of
The
HOMO
of
1
is
slightly
stabilized
(−10.63
eV)
relative
to
that
of
2
(−10.35
eV)
in
agreement
with
1 is slightly
stabilized
(
−
10.63
eV)
relative
to
that
of
2
(
−
10.35
eV)
in
agreement
with
the
experimental
the experimental electrochemical data. The LUMO (−6.95 eV) on the other hand is significantly
the
experimental
electrochemical
LUMO
(−6.95
eV)π-system
the associated
other hand
significantly
destabilized data.
relative
to LUMO
that of 2(data.
(−7.32
eV)
to the
smaller
withisthe
btzpy
electrochemical
The
−
6.95The
eV) due
on the
other
hand
isonsignificantly
destabilized
relative
destabilized
relative
to
that
of
2
(−7.32
eV)
due
to
the
smaller
π-system
associated
with
the btzpy
ligand
to tolterpy
to theπ-system
electron rich
triazole with
moieties.
results
in acompare
larger
to that
of 2 compare
(−7.32 eV)
due to and
the due
smaller
associated
the This
btzpy
ligand
to
ligand
compare
and
due
to the moieties.
electron
rich
triazole
moieties.
This
results inblueagap
larger
HOMO–LUMO
gap electron
for 1 of
3.68
eVtriazole
compared
to that for
2 (3.03
eV) in
mirroring
significantly
tolterpy
and dueto
to tolterpy
the
rich
This
results
a largerthe
HOMO–LUMO
for
shifted
and
emission
HOMO–LUMO
gap for
of
3.68
compared
to that for
2 (3.03
eV) mirroring
the significantly
1 of 3.68
eV absorption
compared
to1 that
foreV
2data.
(3.03
eV) mirroring
the
significantly
blue-shifted
absorptionblueand
shifted
absorption
and
emission
data.
emission data.
Figure 3. Plots of the HOMO (a) and LUMO (b) for the ground state of 1 and the spin density for the
T1 state of 1 (c).
Plots of
of the
the HOMO
HOMO (a)
(a) and
and LUMO
LUMO (b)
(b) for the ground state of 1 and the spin density for the
Figure 3. Plots
state of
of 11 (c).
(c).
T11 state
Molecules 2016, 21, 1382
5 of 12
Molecules
2016, 21, 1382 DFT
Time-dependent
5 of 12
was used to calculate the lowest energy 30 singlet state vertical excitations
at the ground state geometry along with the lowest energy 10 spin-forbidden triplet excitations
Time-dependent DFT was used to calculate the lowest energy 30 singlet state vertical excitations
for 1.at the
The
data state
agree
well with
experimental
spectra
(Supportingtriplet
Information)
with a
ground
geometry
alongthe
with
the lowest energy
10 spin-forbidden
excitationsbut
for 1.
slightThe
overestimation
of
the
energies
of
transitions
compared
to
bands
in
the
UV-visible
absorption
data agree well with the experimental spectra (Supporting Information) but with a slight
spectrum.
The S1 state
is calculated
have an energy
of 2.74
eV (452
nm)UV-visible
and is primarily
HOMO
overestimation
of the
energies of to
transitions
compared
to bands
in the
absorption
1
spectrum.
The Sin
1 state
is calculated
to have
an energy
of 2.74
(452nm)
nm)isand
is primarily HOMO
→
→ LUMO
MLCT
character.
The first
major
transition
(S7eV
, 374
predominantly
composed
of
LUMO
in character.
Theand
first is
major
transition
(S7, 374 character
nm) is predominantly
of a
a HOMO
→1MLCT
LUMO+2
transition
similarly
of 1 MLCT
confirmingcomposed
our experimental
HOMO →
LUMO+2
and isof
similarly
of 1MLCTabsorption
character confirming
experimental
assignment
of the
band transition
in this region
the UV-visible
spectrum.our
The
T1 transition is
assignment
of
the
band
in
this
region
of
the
UV-visible
absorption
spectrum.
The
T
1 transition is
calculated to be at 512 nm (2.42 eV), is of mixed HOMO-2 → LUMO+1 and HOMO-1 → LUMO
calculated to be at 512 nm (2.42 eV), is of mixed HOMO-2 → LUMO+1 and HOMO-1 → LUMO
character and is therefore in agreement with the assignment of the lesser intensity absorptions between
character and is therefore in agreement with the assignment of the lesser intensity absorptions
450 and 550 nm as arising from spin-forbidden direct 3 MLCT 3transitions.
between 450 and 550 nm as arising from spin-forbidden direct MLCT transitions.
The The
lowest
lying
triplet
startingfrom
from
optimized
ground
lowest
lying
tripletstate
stateofof 11 was
was optimized
optimized starting
thethe
optimized
ground
state state
geometry
and
is
calculated
to
lie
2.40
eV
above
the
energy
of
the
ground
state.
The
spin
density
geometry and is calculated to lie 2.40 eV above the energy of the ground state. The spin density was was
plotted
andand
is presented
in in
Figure
unpairedelectron
electron
density
both
the metal
and one
plotted
is presented
Figure3c.
3c.ItItreveals
reveals unpaired
density
on on
both
the metal
and one
of btzpy
the btzpy
ligands
confirmingthe
the 33MLCT
of of
thisthis
T1 state.
Curiously,
unlikeunlike
in the case
of case
of the
ligands
confirming
MLCTcharacter
character
T1 state.
Curiously,
in the
2,
the
T
1
state
of
1
undergoes
a
puckering
like
distortion
of
the
btzpy
ligand
on
which
the
unpaired
of 2, the T1 state of 1 undergoes a puckering like distortion of the btzpy ligand on which the unpaired
electron
density
is localized.
Such distortions
have observed,
been observed,
however,
in theoretical
electron
density
is localized.
Such distortions
have been
however,
in theoretical
calculations
calculations
of
the
T
1 states of bis(tridentate) ruthenium(II) cyclometalated complexes [48] and
of the T1 states of bis(tridentate) ruthenium(II) cyclometalated complexes [48] and [Os(terpy)2 ]2+ [49].
[Os(terpy)2]2+ [49].
Conversion to the chloride salt, [Os(btzpy)2 ]Cl2 (1ClCl ), was achieved by stirring a suspension of 1
Conversion to the chloride salt, [Os(btzpy)2]Cl2 (1 ), was achieved by stirring a suspension of 1
in methanol with Amberlite IRA-400 ion-exchange resin (chloride form) before filtering, removal of
in methanol with Amberlite IRA-400 ion-exchange resin (chloride form) before filtering, removal of
solvent
and and
freeze-drying
from
aqueous
solution.
Removal
couterion was
solvent
freeze-drying
from
aqueous
solution.
Removalofofthe
thehexafluorophosphate
hexafluorophosphate couterion
31 P-NMR
confirmed
by the lack
of the
resonances
in thein19the
F- and
spectra.
19F- and
31P-NMR
was confirmed
by the
lackcorresponding
of the corresponding
resonances
spectra.The
TheUV-visible
UVCl (FigureCl1) in aqueous solution is near identical to that of its analogous
absorption
spectrum
of
1
visible absorption spectrum of 1 (Figure 1) in aqueous solution is near identical to that of its
analogous hexafluorophosphate
salt 1 in acetonitrile.
The complex
is also emissive
in aerated
aqueous
hexafluorophosphate
salt 1 in acetonitrile.
The complex
is also emissive
in aerated
aqueous
solution
with maximum
an emissionatmaximum
at 589slightly
nm, very
slightly blue-shifted
that
of 1 in
with solution
an emission
589 nm, very
blue-shifted
relative torelative
that ofto1 in
acetonitrile.
acetonitrile.
Based on the highly encouraging photophysical data reported above we decided to carry out
Based
on theonhighly
encouraging
photophysical
data1Cl
reported
above
we decided
to carry
out
preliminary
studies
cell uptake
and toxicity.
Complex
was seen
to localise
to the
mitochondria
preliminary studies on cell uptake and toxicity. Complex 1Cl was seen to localise to the mitochondria
in the cancer cell lines HeLa (cervical cancer) and U2OS (osteosarcoma) following a short incubation
in the cancer cell lines HeLa (cervical cancer) and U2OS (osteosarcoma) following a short incubation
time time
of 4 of
h and
with clear phosphorescence seen at concentrations as low as 1 µM. Colocalisation with
4 hours and with clear phosphorescence seen at concentrations as low as 1 μM. Colocalisation
the mitochondrial
stain MitoView
633 was633
seen
under
microscopy
(Figure
4), giving
Pearson0 s
with the mitochondrial
stain MitoView
was
seenconfocal
under confocal
microscopy
(Figure
4), giving
0
correlation
coefficients
r = 0.85 and
for and
HeLa
U2OSand
cells
respectively.
A Pearson
s correlation
Pearson′s
correlationofcoefficients
of r0.7
= 0.85
0.7and
for HeLa
U2OS
cells respectively.
A Pearson′s
coefficient
of 1 indicates
concurrence
the stains, while
indicates
no 0concurrence
correlation
coefficientcomplete
of 1 indicates
completeofconcurrence
of the0 stains,
while
indicates nohence
hencethat
thesecomplex
values indicate
that complexlocalises
1Cl preferentially
localises to the mitochondria.
theseconcurrence
values indicate
1Cl preferentially
to the mitochondria.
Merge
Mitoview 633
U2OS Cells
HeLa Cells
Complex 1 (1μM)
Figure 4. Confocal images of complex 1Cl (green) following 4 h incubation in HeLa and U2OS cells co-
Figure 4. Confocal images of complex 1Cl (green) following 4 h incubation in HeLa and U2OS cells
localised with Mitoview 633 (red) with central overlaid image, scale bars 20 μm.
co-localised with Mitoview 633 (red) with central overlaid image, scale bars 20 µm.
Molecules 2016, 21, 1382
6 of 12
Molecules 2016, 21, 1382
6 of 12
Relative Cell Viability
(cf untreated control)
A
B
1.2
1
0.8
0.6
0.4
0.2
0
0.1
1
10
100
[Complex 1] μM
Clonogenic Survival Fraction
(cf untreated control)
Cl at concentrations up
The cellular
viability
of of
HeLa
cells
withcomplex
complex
The cellular
viability
HeLa
cellsfollowing
following incubation
incubation with
1Cl1at concentrations
up
to 100
waswas
assessed
assay(Figure
(Figure
5).addition,
In addition,
long-term
wasusing
assessed
to µM
100 μM
assessedby
by MTT
MTT assay
5). In
long-term
survival survival
was assessed
> 100
darkin conditions.
This shows
that
at a that
assays
andand
indicated
an LD
usingclonogenic
clonogenic
assays
indicated
an50 LD
100inµM
dark conditions.
This
shows
50 >μM
concentration effective
for for
luminescence
imaging
microscopy
the complex
non-cytotoxic,
lending
at a concentration
effective
luminescence
imaging
microscopy
theiscomplex
is non-cytotoxic,
support
to potential
use asuse
a PDT
in the darkinisthe
desired.
lending
support
to potential
as aagent
PDTwhere
agentnon-toxicity
where non-toxicity
dark is desired.
1.2
1
0.8
0.6
0.4
0.2
0
50
100
[Complex 1] μM
Figure
5. (A)
MTT
and
Clonogenic survival
survival assays
incubation
of HeLa
cells cells
with with
Figure
5. (A)
MTT
and
(B)(B)Clonogenic
assaysfollowing
following
incubation
of HeLa
in the
the dark.
dark. In
mean
andand
standard
deviation
of at of at
increasing
concentrations
complex1Cl
1Clin
increasing
concentrations
of of
complex
Ineach
eachcase,
case,
mean
standard
deviation
3 independent
repeats
is shown.
least least
3 independent
repeats
is shown.
The yield of singlet oxygen generation, φ (1O2), of complex 1Cl was measured in air-equilibrated
The
yield ofagainst
singletthe
oxygen
generation,
φ (1 O2 ), ofAcomplex
1Cl57%
waswas
measured
in air-equilibrated
determined
by direct
acetonitrile
standard
perinaphthenone.
φ (1O2) of
1
acetonitrile
against
the 1Δ
standard
perinaphthenone.
φ (λ
( em
O21275
) of nm)
57%under
was 355
determined
by direct
measurement
of the
g state emission
from 1O2 in theA
NIR
nm irradiation
1 O in the
by a pulsed
laser emission
as described
previously
[50].
It
is
proposed
that
apoptotic
cell
death
after by
measurement
of Nd:YAG
the 1 ∆g state
from
NIR
(λ
1275
nm)
under
355
nm
irradiation
em
2
light
treatment
is
associated
with
localisation
of
photosensitizers
to
mitochondria
[51].
Thus
the
sub- light
a pulsed Nd:YAG laser as described previously [50]. It is proposed that apoptotic cell death after
Cl in the
cellular
localisation,
long-term
survival
following
treatment
of
cancer
cells
with
complex
1
treatment is associated with localisation of photosensitizers to mitochondria [51]. Thus the sub-cellular
dark and the high singlet oxygen yield of 57% indicate the potential for this complex
a
localisation,
long-term survival following treatment of cancer cells with complex 1Cl in the as
dark
and
photosensitizer for PDT theranostic applications. We recognize that the wavelengths of absorption
the high singlet
oxygen yield of 57% indicate the potential for this complex as a photosensitizer for PDT
for 1Cl are at relatively high energy compared to the ideal for a PDT agent andCl
thus are not at the
theranostic
applications.
We recognize that the wavelengths of absorption for 1 are at relatively high
optimum position for maximum tissue penetration for excitation. However, modification of the basic
energy
compared
to the ideal
for a PDT
thus
are not at the optimum
position
for maximum
design
of the complex
by making
the agent
ligandand
more
electron-withdrawing,
for example,
should
be a
tissuerelatively
penetration
for
excitation.
However,
modification
of
the
basic
design
of
the
complex
by
making
easy task. Stabilisation of the LUMO, thus decreasing the HOMO-LUMO gap, would shift
the ligand
more
electron-withdrawing,
for
example,
should
be
a
relatively
easy
task.
Stabilisation
the electronic absorption of the complexes into the desired lower energy region of the spectrum.of the
Based
ondecreasing
these encouraging
results, workgap,
is currently
underway
to fully determine
LUMO,
thus
the HOMO-LUMO
would shift
the electronic
absorptionthe
ofanticancer
the complexes
activity
of 1Cllower
and forenergy
the further
development
of 1Cl and Based
analogues
as a new class results,
of potential
into the
desired
region
of the spectrum.
on thereof
these encouraging
work is
PDT agents.
currently
underway to fully determine the anticancer activity of 1Cl and for the further development
of 1Cl and analogues thereof as a new class of potential PDT agents.
3. Conclusions
3. Conclusions
We have reported the synthesis, characterisation and photophysical properties of a novel
luminescent osmium(II) triazole-based complex. The complex has been shown to exhibit significant
We have reported the synthesis, characterisation and photophysical properties of a novel
quenching of luminescence intensity in the presence of oxygen and a high quantum yield for singlet
luminescent
osmium(II)as
triazole-based
complex.
The
complex
has been in
shown
to exhibittherapy
significant
oxygen sensitization
its chloride salt,
indicating
potential
applications
photodynamic
quenching
of
luminescence
intensity
in
the
presence
of
oxygen
and
a
high
quantum
yield
singlet
as well as luminescence imaging microscopy. The water soluble chloride form of the complexfor
was
oxygen
sensitization
as itscellular
chloride
salt, and
indicating
potential
applications
in photodynamic
therapy
subject
to preliminary
uptake
luminescence
imaging
microscopy
studies. The results
from
studies reveal
that the
complex isThe
successfully
taken up
by twoform
cancerofcell
with was
as well
as these
luminescence
imaging
microscopy.
water soluble
chloride
thelines
complex
mitochondrial
localization
and low
darkand
toxicity.
subjected
to preliminary
cellular
uptake
luminescence imaging microscopy studies. The results
The
use
of
CuAAC
coupling
in
ligand
synthesis opens
upup
diverse
avenues
from these studies reveal that the complex is successfully
taken
by two
cancerfor
celltailored
lines with
derivitisation
and
bioconjugation
that
would
enable
the
optimisation
of
cellular
uptake
and
a wider
mitochondrial localization and low dark toxicity.
scope for organelle targeting within cells. Combined with the attractive photophysical properties, the
The use of CuAAC coupling in ligand synthesis opens up diverse avenues for tailored
complex described in this contribution represents a highly versatile platform for the development of
derivitisation and bioconjugation that would enable the optimisation of cellular uptake and a wider
dual-mode luminescence imaging/singlet oxygen sensitisation photodynamic theranostic complexes.
scope for organelle targeting within cells. Combined with the attractive photophysical properties, the
complex described in this contribution represents a highly versatile platform for the development of
dual-mode luminescence imaging/singlet oxygen sensitisation photodynamic theranostic complexes.
Molecules 2016, 21, 1382
7 of 12
Plans to pursue these studies are in progress and results from these studies will be published elsewhere
in due course.
4. Experimental Section
4.1. General Methods
Ammonium hexachloroosmate(IV) was purchased from Alfa Aesar (Ward Hill, MA, USA) whilst
all other reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), Acros Organics
(Thermo Fisher Scientific, Geel, Belgium) or Fluorochem (Hadfield, UK) and used as supplied.
The ligand btzpy [37] and complex 2 [52] were prepared by literature procedures. NMR spectra
were recorded on a Bruker Ascend 400 MHz spectrometer (Billerica, MA, USA), with all chemical shifts
being quoted in ppm referenced relative to the residual solvent signal (MeCN, 1 H: δ = 1.94, 13 C: 1.32,
118.26; DMSO, 1 H: δ = 2.50, 13 C: 39.52 ). High-resolution mass spectrometry was performed on an
Agilent 6210 TOF instrument (Santa Clara, CA, USA) with a dual ESI source. UV-visible absorption
spectra were recorded on an Agilent Cary 60 spectrophotometer whilst emission spectra were recorded
on a Fluoromax 4 spectrophotometer (aerated and degassed in acetonitrile and data at 77 K in a
4:1 EtOH/MeOH glass). Lifetime measurements were performed using an Edinburgh instruments
Mini-Tau spectrometer (Edinburgh, UK). Emission quantum yields (φem ) were measured for degassed
MeCN solutions, with degassing carried out via three repeat freeze-pump-thaw cycles. Quantum
yields are quoted relative to [Ru(bpy)3 ][PF6 ]2 in aerated MeCN, with analyte solutions being excited
at a single wavelength at a point of common optical density. Thus, φem values are determined from
the ratio of integrated area under the corrected peaks, with an assumed experimental uncertainty of
±20%. Cyclic voltammograms were recorded using an Autolab PGSTAT100N potentiostat with NOVA
electrochemical software (version 1.10.1.9). Analyte solutions were prepared using nitrogen saturated
dry acetonitrile, freshly distilled from CaH2 . All measurements were conducted at room temperature
under a stream of dry nitrogen at potential scan rates ranging from 20 to 500 mV·s−1 . [NBu4 ][PF6 ]
was used as a supporting electrolyte, being recrystallised from ethanol and oven dried prior to use,
with a typical solution concentration of 0.2 mol dm−3 . The working electrode was a platinum disc,
with platinum wire utilised as the counter electrode. The reference electrode was Ag/AgCl, being
chemically isolated from the analyte solution by an electrolyte containing bridge tube tipped with a
porous frit. Ferrocene was employed as an internal reference, with all potentials quoted relative to the
Fc+ /Fc couple
4.2. Synthesis of [Os(btzpy)2 ][PF6 ]2 (1)
Ammonium hexachloroosmate(IV) ([(NH4 )2 OsCl6 ], 150 mg, 0.341 mmol) and 2.5 equivalents of
2,6-bis(1-phenyl-1H-1,2,3-triazol-4-yl)pyridine (310 mg, 0.85 mmol) in ethylene glycol (25 cm3 ) was
heated at reflux overnight under nitrogen. The resulting mixture was then allowed to cool to room
temperature. 2.5 equivalents of aqueous NH4 PF6 was added resulting in a dark brown precipitate
which was collected by filtration and washed with cold water and diethyl ether. This was then
redissolved in acetonitrile, cooled in the fridge overnight and filtered to remove unreacted ligand.
The solvent was removed from the filtrate and the residue recrystallized from dichloromethane/hexane
to give an orange powder. Yield = 185 mg, 45%; 1 H-NMR (400 MHz, CD3 CN): δ 9.13 (s, 4H); 8.36
(d, J = 8.0 Hz, 4H); 8.01 (t, J = 8.0 Hz, 2H); 7.49–7.60 (m, 20H). 13 C-NMR (101 MHz, CD3 CN): δ 153.26,
151.74, 138.26, 136.94, 131.12, 131.07, 124.98, 121.64, 120.18. ESI HRMS: calculated for [C42 H30 N14 Os]2+
m/z = 461.1191; found m/z = 461.1195.
4.3. Synthesis of [Os(btzpy)2 ]Cl2 (1Cl )
A suspension of 1 (100 mg, 0.083 mmol) in methanol (25 cm3 ) was stirred with Amberlite IRA-400
ion-exchange resin (chloride form, 200 mg) for 24 h at R.T. in the dark. The resin was removed by
filtration and the solvent then removed by evaporation. The residue was then dissolved in water and
Molecules 2016, 21, 1382
8 of 12
the solution was freeze dried to yield 1Cl as an orange powder. Yield = 71 mg, 87 % 1 H-NMR (400 MHz,
d6 -DMSO): δ 10.15 (s, 4H); 8.58 (d, J = 8.0 Hz, 4H); 8.21 (t, J = 8.0 Hz, 2H); 7.66–7.72 (m, 8H); 7.47–7.57
(m, 12H). 13 C-NMR (101 MHz, d6 -DMSO): δ 152.01, 150.32, 137.57, 135.58, 130.08, 130.00, 125.27, 120.46,
119.31. ESI HRMS: calculated for [C42 H30 N14 Os]2+ m/z = 461.1191; found m/z = 461.1192.
4.4. Computational Details
The geometries of cations for complexes 1 and 2 were optimized using DFT calculations at the
B3LYP [53,54] level of theory (20% Hartree-Fock). Phenyl substituents of the btzpy ligand were
simplified to methyl groups to reduce computational cost. The Stuttgart-Dresden relativistic small
core effective pseuopotential and basis set was used for osmium [55] and 6-311G* basis sets used for
all other atoms [56]. Optimised minima were confirmed through vibrational frequency calculations.
TDDFT calculations were carried out at the optimized ground state geometries to compute the vertical
excitation energies (lowest 30 singlet and 10 triplet roots) and hence the simulated optical absorption
spectra. The T1 states were also optimized and the spin density calculated and plotted. All calculations
were carried out using the NWChem 6.6 software package [57] with geometries, molecular orbital
surfaces and spin densities viewed and plotted using the ECCE graphical user interface.
4.5. Cell Culture
Both HeLa (human cervical cancer) and U2OS (human bone osteosarcoma) cell lines were
purchased from American Type Culture Collection–LGC partnership (Teddington, UK) and used
within 20 passages of purchase. Cells were cultured in Dulbecco0 s modified Eagles Medium (DMEM)
(Lonza, Cambridge, UK) with 10% fetal calf serum (FCS) (Lonza, Cambridge, UK) and incubated at
37 ◦ C under 5% CO2 . Both cell lines were routinely checked for mycoplasma infection. Complex 1Cl
was stored as a stock solution at 10 mM in DMSO.
4.6. Luminescence Imaging and Colocalisation Studies
Cover glasses (22 × 22 mm) were sterilised (industrial methylated spirits, IMS) and placed flat
in 6-well plates. Cells were seeded at a density of ~1 × 105 cells per well and allowed to adhere
overnight in culture media. Complex 1Cl (1 µM) was added and incubated for 4 h, for co-localisation
studies MitoView™ 633 (Biotium) was added for the final 15 min, prior to cells being washed 3 times
in PBS and fixed in 4% paraformaldehyde solution in PBS at 4 ◦ C for 20 min. Following a further wash
(PBS × 3) the coverslips were mounted to microscope slides (IMMU-MOUNT™, Life Technologies Ltd.,
Paisley, UK). The slides were imaged by confocal microscopy (Nikon A1 confocal) using a 60× lens
(CFI Plan Apochromat VC 60× oil, NA 1.4). An argon laser (405 nm and 561 nm) was used to excite
complex 1Cl and a diode laser (642 nm) was used to excite MitoView™ 633. Colocalisation indices were
calculated using the open source imaging software Fiji (based on ImageJ) and the coloc 2 colocalisation
tool. The threshold regression chosen was Bisection.
4.7. Cell Viability Assay-MTT
96-well plates were seeded with HeLa cells at 1000/well and incubated overnight. Wells were
treated with 0.1–100 µM complex 1Cl or DMSO control and incubated for 4 h before replacing with
fresh media. After 5 days further growth, 25 µL of 3 mg cm−3 thiazoyl blue (MTT) solution was added
to each well. Following incubation for 3 h the solution was removed from each well and 250 µL/well
DMSO added ensuring mixing of crystals. Optical density of wells at 540 nm was recorded on a plate
reader (Multiskan fc, Thermo Fisher Scientific, Warrington, UK).
4.8. Clonogenic Survival
Six-well plates were seeded with HeLa cells at 400 cells/well and incubated overnight. Wells were
treated with DMSO, 50 µM or 100 µM complex 1Cl for 4 h before replacing with fresh media. Plates were
Molecules 2016, 21, 1382
9 of 12
incubated for 8–10 days to form colonies before staining with 4% methylene blue in 70% methanol
and counting. Each colony was considered to represent a single surviving cell and survival fraction
calculated for each condition compared to DMSO control.
Supplementary Materials: The following are available online at www.mdpi.com/1420-3049/21/10/1382/s1,
Figure S1: 1 H-NMR spectrum of 1 (CD3 CN), Figure S2: 13 C-NMR spectrum of 1 (CD3 CN), Figure S3: ESI mass
spectrum of 1, Figure S4: 1 H-NMR spectrum of 1Cl (d6 -DMSO), Figure S5: 13 C-NMR spectrum of 1Cl (d6 -DMSO),
Figure S6: ESI mass spectrum of 1Cl , XYZ coordinates for the optimized geometries of the ground and lowest lying
triplet states of 1, Figure S7: Time-dependent DFT UV-visible absorption spectrum of 1, Table S1: Summarised
time-dependent DFT data for 1.
Acknowledgments: The authors thank the State of Libya (SAEO), University of Huddersfield (PAS), Yorkshire
Cancer Research, Cancer Research UK, BBSRC, The Wellcome Trust, EPSRC KTA and University of Sheffield
(LKM, HEB & JAW) for supporting this work. Microscopy was performed on equipment purchased on the
following grants: Welcome Trust grant WT093134AIA and MRC SHIMA award MR/K015753/1. As a member of
the UK Materials Chemistry Consortium PIPE acknowledges the EPSRC (EP/L000202) and the UK HPC national
resource, Archer, as well as the University of Huddersfield High Performance Computing Research Group for
computational facilities utilized in this work.
Author Contributions: SAEO carried out the majority of experimental synthetic and spectroscopic work.
PAS carried out additional experimental work and acted as co-supervisor for SAEO. LKM carried out cellular
uptake and imaging studies under the supervision of HEB and JAW. Principal investigator PIPE carried out
computational calculations and was the main manuscript author, and PAS, HEB, LKM and JAW assisted in writing
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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